Flashcards in Molecular techniques and diagnosis Deck (92):
What type of molecule is run on a Southern Blot?
What type of molecules is run on a Northern Blot?
What type of molecule is run on a Western Blot?
What type of bonds to restriction enzymes digest?
What are restriction enzymes?
Enzymes that recognise and cut specific DNA sequences (restriction sites)
What is of note about the restriction site sequences and the way they are cut?
Mostly palindromes of 4,5,6,8 bases and mostly the cuts leave 'sticky ends' but can also produce 'blunt ends'
What are endonucleases' natural function in bacteria?
To recognise and degrade foreign DNA
Why do bacteria not digest their own DNA with the endonucleases they produce?
Their own DNA is protected by methylation
What is run with the test DNA on a gel during DNA gel electrophoresis so that the size of the bands of DNA can be calculated?
Reference DNA markers of known sizes
How can DNA be visualised on the agarose gel?
By staining e.g. with ethidium bromide which stains DNA by intercalating between its bases
What are the four requirements for gel electrophoresis?
1. Gel - gel that allows separation of DNA fragments
2. Buffer - allows charge on the DNA samples across the gel
3. Power supply - generates charge difference across the gel
4. Stain/detection - to identify the presence of the separated DNA
Why use restriction analysis?
1. Investigate size of fragments - small indel?
2. Investigate mutations - at DNA level (change RE site?)
3. Investigate DNA variation - e.g. DNA fingerprinting
4. To clone DNA - take DNA length of interest after RE and ligate it into a vector
What is the function of DNA ligase?
It forms new phosphodiester bonds
Why can sticky ends created by different restriction enzymes sometime hybridise?
The restriction enzymes recognise different sequences but create the same complimentary sticky ends
When sticky ends created by different restriction enzymes are joined together by hybridisation and ligase, what is of note about their restriction site?
The restriction sites are not re-formed, as they are each the product of a RE which recognised a different site so are a mix of the two sites
What are plasmids?
Small circular dsDNA found in bacteria (mini-chromosomes), that carry genes to replicate independently. They can transfer to other bacteria
How is a recombinant vector introduced into bacterium?
Explain the steps of gene cloning
1. Plasmid and DNA of interest are cut with REs leaving complimentary sticky ends
2. Plasmid and DNA sticky ends hybridise and a phosphodiester bond is formed by DNA ligase
3. Insertion of the DNA of interest into the Multiple cloning site (polylinker) of the plasmid interrupts the LacZ gene on the plasmid, a gene responsible for blue colour
4. The plasmids are mixed with the bacteria and some take them up by a process called transformation
5. Antibiotics can be added to sort out which of the bacteria contain the plasmid (it carries an antibiotic resistance gene) and which have no plasmid
6. The remaining bacteria contain plasmids and can be sorted by colour. Bacteria which have taken up a plasmid that does not contain the DNA of interest turn blue. Non-blue bacteria can then be isolated
7. These isolated bacteria can then be allowed to reproduce - they are now clones which all contain the DNA of interest
Why clone human genes?
1. To make useful proteins e.g. insulin
2. To find out what genes do - structure, expression, control
3. Genetic screening e.g. HD etc...
4. Gene therapy? e.g. cystic fibrosis
How was the mammalian proinsulin gene transformed into a bacterium?
1. proinsulin mRNA converted to DNA using reverse transcriptase
2. RE cut and ligated into a plasmid -> recombinant plasmid
3. Transformed into E.coli or mammalian cells (these are better because they give the correct post-translational modifications)
Why is PCR used?
1. To amplify a specific target DNA sequence
2. To investigate single base pair mutations
3. To investigate small deletions or insertions
4. To investigate variation, genetic relationships - DNA profiling, DNA typing
What are the three different temperature cycles used in PCR?
Denature - 95oC
Anneal - 60oC
Polymerise - 72oC
What type of DNA polymerase is required for PCR
Thermostable DNA polymerase e.g. Taq polymerase
How does PCR work?
Reaction mixture contains: forward and reverse primers, lots of dNTPs, thermostable DNA pol.
1. Denature DNA at 95oC
2.Cool to 60oC to anneal primers
4. DNApol extends DNA from primer at 72oC
How can proteins be separated by protein gel electrophoresis?
size, shape or charge
What is the difference in the orientation of a protein gel electrophoresis as compared to DNA gel electrophoresis?
Protein gel is vertical (wells at top) - anode/cathode direction depends on the protein
DNA gel is horizontal - anode opposite wells
What are the different types of protein gel electrophoresis?
1. Native folding
2. SDS-PAGE - unfolded
3. isoelectric focusing
How does SDS-PAGE separate proteins?
SDS binds to proteins, denaturing their 3D structure and giving them a uniform negative charge. Proteins are therefore separated purely on the basis of their size
How does isoelectric focusing (IEF) separate proteins?
0The gel has a stable pH gradient when an electric field is applied. Therefore when proteins are added they migrate to the pH where there isoelectric point (pI is, at which point they have no net charge and stop migrating. Therefore proteins are separated by their pI
How are proteins separated by 2D-PAGE?
Two steps of separation, perpendicular to each other:
1. IEF - separates by pI
2. SDS-PAGE separates by size
What is the advantage of using SDS-PAGE?
Allows the separation of complex mixtures of proteins. Important for diagnosing disease states in different tissues
How can you identify a protein?
1. Digest protein with trypsin
2. Perform mass spectrometry
3. Generate list of peptide sizes
4. Use database of predicted peptide sizes for known proteins to:
5. Identify protein
Analysis of all proteins expressed from the genome
Define molecular diagnosis
Analysis of a single purified protein
How does enzymatic cleavage of proteins differ from DNA cleavage by REs?
They are effectively the protein equivalent of REs, though there are not as many of them
What are the two broad substances used to specifically cleave proteins?
Part of an antigen (few amino acids on a protein) that is recognised by the immune system (antibodies, B cells or T cells)
What are polyclonal antibodies?
Multiple different antibodies produced by many B lymphocytes that are specific to one antigen (but multiple epitopes)
How do you produce polyclonal antibodies in the lab?
Inject a mouse with antigen 3-4 times at two week intervals, then bleed the mouse and collect the antigen-specific antibodies
What are monoclonal antibodies?
Identical antibodies produced by one B lymphocyte that are specific to one antigen (and one epitope)
How do you produce monoclonal antibodies in the lab?
1. Inject a mouse with an antigen.
2. Isolate spleen cells and fuse with continuously dividing culture myeloma cells
3. Select and grow hybrid cells
4. Select cells making antibody of desired specificity
5. Either induce tumours in a mouse and collect antibody or grow in mass culture and collect antibody
What is Western blotting?
When you make a nitrocellulose replica of a gel electophoretogram and incubate it with:
1. Primary antibody to bind to protein of interest
2. Secondary antibody to detect where primary antibody is bound e.g. enzyme that digests substrates to produce a colour that can be detected
What is Enzyme-linked immunoabsorbent assay (ELISA)?
A technique to measure the concentration of proteins in solution
How does ELISA work?
1. Bind antigens to surface of a well
Which important plasma hormones can be measured using ELISA?
In non-ELISA enzyme assays, how can the product of an enzymatic reaction be measured (and thus the concentration of enzyme present)?
Spectrophotometery - absorbance of light
Why is the measurement of enzymes important?
1. Metabolic disorders
2. Diagnosis of disease - serum enzymes
Name three clinically important serum enzymes that are markers for liver damage/ disease
Aspartate transaminase (AST)
Alanine transaminase (ALT)
Name a clinically important serum enzyme that is a marker for pancreatitis
Name a clinically important serum enzyme that is a marker for bone disorders (see in some bone cancers)
What is the gold standard for diagnosis of MI?
Measurement of cardiac troponin (cTnI) by ELISA - quick diagnosis
What isoenzyme is specific to myocardium, and is a marker for MI?
Creatine kinase-MB (CK-2)
How can the concentration of important metabolites be measured?
Using enzymes e.g. glucose oxidase can be used to measure the glucose present in blood on a test strip. Glucose oxidase catalyses a reaction which converts glucose into H2O2 (amongst other things) which then converts the test strip to a coloured dye that is measured by the glucose monitor
Explain DNA hybridisation
the process of combining two complementary single-stranded DNA or RNA molecules and allowing them to form a single double-stranded molecule through base pairing (hydrogen bonding between bases)
What is meant by DNA denaturing?
When dsDNA is heated (or treated with alkaline solution) which breaks the hydrogen bonds between bases, releasing ssDNA
What is meant by DNA renaturing/ annealing?
When ssDNA upon cooling (or pH=7) comes back together into dsDNA by reforming hydrogen bonds between complimentary bases
What is a DNA probe?
A piece of ssDNA which is labelled with a radioactive or fluorescent marker and which has an identical sequence to a target sequence of DNA
How are radioactive probes detected?
autoradiography: image on an X-ray film
What is Southern Blotting?
A technique which uses DNA probes to identify complementary DNA sequences after gel electrophoresis
What is Northern Blotting?
A technique which uses DNA probes to detect RNA sequences after gel electrophoresis
What is Western Blotting?
In a similar way to nucleic acid blotting, protein is separated by protein gel electrophoresis and detected using antibodies
Describe the process of Southern Blotting
1. Digest DNA using restriction enzyme(s)
2. DNA gel electrophoresis - separate fragments
2. Transfer DNA onto a nylon
3. Hybridisation of a probe - specific to a piece of DNA
4. Visualisation of labelled probe - e.g. exposure of filter to X-ray film
What are some of the sources of the DNA used in Southern Blotting?
Why are probes necessary to visualise DNA gel electrophoresis - why can't we visualise DNA with just stains?
It allows the identification (presence or absence) of specific sequences of DNA to be picked out of large smear of many many different pieces of genomic DNA on a gel electrophoresis
In Southern blotting when transferring DNA from a gel to a solid support, what is the purpose of the alkaline solution?
It denatures the dsDNA into ssDNA which are then transferred onto the membrane
In Southern Blotting how is DNA transferred from the gel onto the nylon or nitrocellulose filter?
1. Blotting by capillary action onto the gel (stack of paper towels onto of filter soak up alkaline solution towards them - moving DNA at the same time)
2. Or electrophoretic transfer - voltage is applied across the set-up
In what form is the DNA on the nitrocellulose/nylon filter?
How are DNA probes made?
1. Cloned piece of DNA already available
2. Or produced in lab using an oligonucleotide synthesiser - used dNTPs to synthesis oligos programmed into the machine
In general why do we use Southern Blotting?
1. Detect pieces of DNA from complex mixtures
2. Detect very small pieces of DNA that may not be visible by staining DNA on a gel
3. Many cases we use it along with PCR to detect things like gene structure (large deletions or duplications), gene expansion and repeats (fragile X syndrome), mutations (using allele-specific probes) and genetic variation (DNA fingerprinting)
List the three characteristics of DNA probes in blotting
1. Probes do not have 100% similarity to the target sequence
2. Probes do not completely align with the target sequence (they still work if only attach by their end)
3. Probes do not affect the position of the target sequence on a gel
What is another name for the Sanger chain termination method?
Dideoxy chain termination method
What is the difference between dNTP and ddNTP?
Dideoxynucleotide triphosphate has a H at its carbon 3 position, not an -OH group, therefore DNApol cannot add a dNTP to it (no phosphodiester bond can be formed). ddNTP incorporation into an elongating DNA strand will therefore block further elongation
Explain how the Sanger chain termination method is used to sequence DNA
There are four tubes for reactions. In each tube there is a mixture of DNApol, labelled primer, all the dNTPs (dATP,dCTP,dGTP and dTTP) and also one of the ddNTPs (e.g. ddCTP).
We incubate them at 37oC and from each tube we get a mixture of new DNA molecules of different lengths depending on where they have incorporated the ddNTP molecule. At each corresponding base either:
1. a dNTP can be added and the chain will continue growing
2. a ddNTP can be added and the chain stops growing
These tubes are then run on an electrophoresis gel (one well per tube) and the sequence of bases can be read from the smallest fragment(closest to the anode) to largest fragment (closest to the cathode)
How has Sanger sequencing been modified in fluorescent dideoxy DNA sequencing?
The reaction is now done in the same tube using fluorescently labelled ddNTPs. The fragments are then run on a very thin capillary gel and detected as they fall off the end by a laser. This produces a computer-generated chromatogram from which can read off the sequence directly.
Ethical considerations of DNA sequencing
1. Who would be interested in your genome info?
2. Can the knowledge help prevent illness later in life?
3. Does it open up areas for discrimination?
4. Who owns DNA sequence?
What is a heteroduplex?
A heteroduplex is a double-stranded nucleic acid in which each complementary strand has come from a different source (e.g. genomic and probe strands of DNA)
How can the use of allele-specific probes in PCR be used to detect SNPs?
They can be designed so that it will bind to an A base at its 3' end but not an T base e.g. the normal haemoglobin sequence but not the sickle cell sequence. The binding of the 3' end of a probe is particularly important as this has to be tightly bound for the DNApol to extend it
Why do you have to be more careful of the conditions with Northern blotting as compared to Southern blotting?
RNA is less stable than DNA and therefore degrades more easily
How does reverse transcriptase PCR (RT-PCR) differ from normal PCR?
1. mature mRNA is converted to cDNA by reverse transcriptase and a primer which binds to the polyA tail
2. RNAse is then added - a nuclease which specifically degrades RNA
What do microarrays allow us to do?
Analyse 1000s of genes simultaneously in a 'genome-wide' analysis
How can microarrays be used to study conditional gene expression?
1. Isolate mRNA from cells of interest and reference normal cells
2. Convert to cDNA using reverse transcriptase and differently labelled primers
3. Combine targets in equal quantities and hybridise to specific oligonucleotides on the microarray
4. Detect comparative fluorescence from each
How does array comparative genome hybridisation (aCGH) work?
1. Extract DNA from cell of patient and normal control cells and chop it up.
2. Label with two different fluorochromes
3. Mix in equal quantities and hybridise to microarray of clones of oligonucleotides
4. Detect fluorescence and analyse ratio for each cell - align to database of clones
Why use array technology?
1. Investigate 1000s of genes simultaneously
2. Investigate chromosome deletions/ duplications (aCGH)
3. Investigate conditional gene expression
What is another name for DNA fingerprinting?
What is the basis of DNA fingerprinting?
That individuals have 'minisatelites' (10-60bp) which show copy number variation and therefore are highly variable and different between individuals
How is DNA profiling different these days?
1. Looks at 16 variable regions using smaller repeats called small tandem repeats (STRs)
2. Uses PCR to amplify
What are the uses of DNA profiling?
Forensics - due to PCR only need small amounts of DNA
What is karyotyping?
The analysis of a complete set of chromosomes of an individual
Why is FISH useful?
Can locate specific DNA sequences INSIDE cells
What is chromosome painting?
A technique which uses a family of probes that are all specific to sequences that are present on only chromosome e.g. 1. A computer can recognise the wavelengths of probes and make each family an artificially bright colour, so individual chromosomes are more obvious`
Why use FISH?
1. Investigates genes in situ - deletion, duplication, translocation
2. Investigate chromosome structure - del, dup or trans
3. Investigate chromosome number - e.g. chromosome painting
4. Investigate chromosome behaviour - e.g. anaphase lag